Coculture of native human acute myelogenous leukemia blasts with fibroblasts and osteoblasts results in an increase of vascular endothelial growth factor levels

Abstract:  Objectives : Angiogenesis seems important in the development of acute myelogenous leukemia (AML). Proangiogenic vascular endothelial growth factor (VEGF) is constitutively secreted by the AML blasts for a subset of patients, but it can also be released by non-leukemic bone marrow cells. Methods : VEGF levels were determined after coculture of native human AML blasts with fibroblast lines, osteoblastic sarcoma cell lines, normal bone marrow stromal cells and normal osteoblasts. Cultures were prepared with leukemic and non-leukemic cells separated by a semipermeable membrane or in direct contact. Results : The non-leukemic cells usually showed higher spontaneous VEGF release than AML cell populations. Coculture of AML blasts with HFL1 fibroblasts caused a supra-additive increase of VEGF levels when the cell populations were cultured separately, and the increase was also observed when cells were cultured in direct contact. An increase was also observed when AML blasts were cultured with osteoblastic sarcoma cells, normal bone marrow stromal cells and normal osteoblasts. Coculture had divergent effects on VEGF mRNA levels both for leukemic and non-leukemic cells, but increased mRNA levels were commonly observed especially for the non-leukemic cells. Cytokine inhibition experiments suggest that IL1 is important for the VEGF-increasing crosstalk, whereas the mechanisms are probably heterogeneous for coculture with osteoblasts. Conclusion : The bi-directional crosstalk via local cytokine networks between AML blasts and non-leukemic cells results in increased local VEGF levels, an observation suggesting that VEGF-targeting antiangiogenic therapy should be considered as a general therapeutic strategy in AML.     

Relationship between patients' age, bone marrow karyotype, and outcome of induction therapy in acute myelogenous leukemia

Bone marrow karyotypes were performed in 88 cases of adult acute myelogenous leukemia (AML) at diagnosis and classified NN (normal), AA (abnormal), and AN (mixture of normal and abnormal metaphases). A clear relationship was found between karyotype and complete remission (CR) rate: 58% CR in (NN + AN) cases; 14% CR in AA cases (P less than .009). This relationship was even stronger when only patients under 60 years of age were studied. Considering failures of induction treatment, no relationship was found between the NN/AN/AA classification and a drug resistance. In patients over 60, the worse prognosis could be explained by an inferior ability to tolerate intensive treatment.     

慢性粒细胞白血病急变为急性巨核细胞白血病1例

急性巨核细胞白血病(AMLK)占所有急性髓系白血病中发生率的0.5%~1.2%,其中慢性粒细胞白血病(CML)转化为AMLK的比例报道不一。现报道1例以骨髓纤维化(MF)首诊的AMLK,最终通过电镜、免疫分型以及分子生     

Primary human acute myelogenous leukemia cells release matrix metalloproteases and their inhibitors: release profile and pharmacological modulation

Objectives: Angiogenesis seems important for both leukemogenesis and chemosensitivity in acute myelogenous leukemia (AML). Angiogenesis is regulated by the balance between pro‐ and antiangiogenic cytokines, which also indicates an important role of matrix metalloproteases (MMPs) and their natural inhibitors, tissue inhibitors of metalloproteases (TIMPs). We investigated the constitutive release of MMPs and TIMPs for a large group of consecutive AML patients. Methods: AML cells were cultured in vitro either alone or together with microvascular endothelial cells, and levels of MMPs and TIMPs were determined in culture supernatants. Results: AML cells showed constitutive release of several MMPs and TIMPs. For all patients, detectable MMP‐10 release was observed, and most patients showed detectable release of at least one additional MMP, usually MMP‐9 or MMP‐2. A significant correlation was found between MMP‐9 and TIMP‐1 release and the release of several CCL and CXCL chemokines. MMP‐9 release was higher for AML cells with monocytic differentiation corresponding to the FAB‐subtype M4/M5 AML; it was mainly released in its inactive form, but endogenously active MMP‐9 could be detected even in the presence of the constitutively released TIMP‐1/2. Endothelial cells released relatively high levels of MMP‐10, and these levels were further increased by coculture with AML cells. Patients achieving complete hematological remission after only one induction cycle showed relatively low constitutive MMP‐2 release. Conclusion: We conclude that primary human AML cells show constitutive release of both MMPs and TIMPs, and this release may be important for leukemogenesis and possibly also for chemosensitivity.     

Flt3-L是否为体外制备急性髓系白血病患者DC瘤苗的更佳选择?

目的:通过对体外使用Flt3-L(FL)及粒-巨噬细胞集落刺激因子(GM-CSF)诱导完全缓解期急性髓系白血病(AML-CR)患者的骨髓中单个核细胞(BMNC)分化为树突状细胞(DC)、分泌白介素(IL-12P70),干扰素-α(INF-α)的情况,及所诱导DC体外刺激自体T细胞(auto-T cells)活化增殖能力的观察,进一步了解FL在DC瘤苗制备中的作用特点。方法:分离AML-CR患者BMNC采用FL／GM-CSF单因子培养于RPMI1640完全培养基中至第8天,加入LPS刺激活化后流式细胞仪明确DC免疫表型,ELISA法测定培养液上清中IL-12P70、INF-α,收集阳性细胞体外激发增埴auto-T细胞,MTT法测试T细胞增殖情况。结果:10例AML患者的BMNC经FL／GM-CSF分别培养至第72 h均开始有细胞成簇生长,流式细胞检测显示CD1a、CD83、CD80、CD86、HLA-DR均表达明显上调,ELISA测定FL组IL-12P70,INF-α分泌量较GM-CSF组有显著增加。2组均可产生较强的T细胞增殖效应,与GM-CSF比,FL组对CD4 T细胞的增殖作用更强。结论:在体外,FL可更强诱导AML-CR患者BMNC产生DC,同时分泌大量IL-12P70及INF-α。故与GM-CSF相比,FL可能具有更佳的抗肿瘤免疫作用。     

Treatment of refractory undifferentiated acute myelogenous leukemia with all-trans-retinoic acid

A patient is described with undifferentiated acute myeloblastic leukemia refractory to two courses of daunorubicin and cytosine arabinoside. Because some the myeloblasts developed morphologic features of promyelocytes, the patient was treated with all-trans-retinoic acid (ATRA) in an attempt to promote maturation. Cytogenetic studies and sensitive molecular analysis did not reveal any abnormality classically associated with acute promyelocytic leukemia. Serial bone marrow biopsies demonstrated myeloid maturation, and the patient uneventfully went into a sustained complete remission. A review of the literature confirms this to be an apparently hitherto undescribed response to ATRA that may have therapeutic implications in similar patients. © 1994 Wiley-Liss, Inc.     

Lipoteichoic acid derived from Enterococcus faecalis modulates the functional characteristics of both normal peripheral blood leukocytes and native human acute myelogenous leukemia blasts

OBJECTIVES: Several case reports have described complete hematological remissions for patients with otherwise untreated acute myelogenous leukemia (AML) who receive hematopoietic growth factor therapy during complicating bacterial infections. This may be caused by indirect cytokine effects, but direct effects of infecting agents on the malignant cells are also possible because bacterial molecules can bind to specific receptors expressed by normal and malignant leukocytes. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria, and it can activate normal immunocompetent cells through binding to specific cell membrane receptors. METHODS: We investigated effects of LTA derived from Enterococcus faecalis on in vitro cultured (i) normal peripheral blood mononuclear cells (PBMC); (ii) remaining T cells derived from patients with hematologic malignancies and chemotherapy-induced leukopenia; and (iii) native human AML cells. RESULTS: Increased interleukin 1beta (IL1beta) and IL8 release by in vitro cultured normal PBMC was observed after stimulation with LTA at concentrations > or =5 microg/mL; these levels were lower than for lipopolysaccharide (LPS)-stimulated cells and LTA antagonized LPS-induced cytokine release by normal PBMC. In most cases LTA did not alter T-cell proliferation for patients with chemotherapy-induced leukopenia. The LTA effects on AML blasts were investigated for 62 consecutive patients. LTA altered either cytokine (granulocyte-macrophage colony-stimulating factor + stem cell factor + IL3)-dependent proliferation or the release of IL1beta/IL8 for 23 patients; the effects were divergent but increased proliferation/cytokine levels were most commonly observed. CONCLUSION: The LTA derived from E. faecalis can modulate the functional characteristics of normal leukocytes and native human AML blasts.     

老年急性髓系白血病的治疗现状

急性髓系白血病（AML）随着年龄的增长发病率逐渐升高。由于老年AML患者化疗完全缓解率低,治疗相关死亡率高,长期生存率低,预后差,尚缺乏统一有效的治疗策略。本文就目前老年AML治疗现状作简单综述,探讨传统化疗、造血干细胞移植及新的靶向药物在老年AML治疗中的应用。     

Conservative treatment for patients over 80 years with acute myelogenous leukemia

In order to evaluate the best treatment of very elderly patients with AML, we have retrospectively analyzed 60 cases of patients aged more than 80 years, with a diagnosis of AML and observed from January 1988 to December 1998. Six of these patients were subsequently referred to other centers; of the remaining 54 patients, 20 (37%) received only supportive care, whereas 34 (63%) required palliative chemotherapy to control leukocytosis, after a median time from diagnosis of 9 days (range 0-253). Median overall survival was 13 weeks (range 1-105): 21 (39%) and 6 (11%) patients survived more than 6 and 12 months, respectively. Twenty-eight patients (51.8%) died from progressive disease, 19 (35.1%) died from AML-related or unrelated causes in the phase of stable disease, while in 7 patients the cause of death was unknown. In univariate analysis, PS > 2 and WBC > 50 x 10(9)/L had an adverse prognostic significance on survival. Our results, as compared with those reported in the literature for patients over 80 years treated with intensive chemotherapy, support the idea that intensive chemotherapy is usually not indicated in very elderly patients with AML, and that conservative treatment and the primary strategy of "watch-and-wait" presently seems to be the best choice.     

Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts

We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).     

The EORTC trials for acute myelogenous leukemia

Since the early 80s, the EORTC Leukemia Group has included 3947 patients in 8 consecutive phase III studies on acute myelogenous leukemia, with stratification according to the age groups. Some of these patients were included by other cooperative groups (GIMEMA, HOVON), within intergroup studies. The main hypotheses tested by randomization were intensive chemotherapy consolidation, allogeneic and autologous bone marrow transplantation in the younger patients. In patients aged 60 years or more, a wait and see policy followed by a palliative chemotherapy was compared to an immediate conventional induction treatment, mitoxantrone was compared to daunorubicin, and, during remission, a low dose Ara-C maintenance treatment to no maintenance. The main results can be summarized as follows: Allogeneic BMT, and, relatively, autologous BMT do better than intensive chemotherapy consolidation with regards to the disease-free survival, but not for the overall survival after remission. In the elderly, an immediate induction and a low-dose Ara-C maintenance treatment are preferable to the other options tested. Mainly in patients aged more than 45, the supplementary toxicity of more intensive chemotherapy consolidation, with high dose Ara-C, counter balanced the increased anti-leukemic effect. Finally the trials randomizing GM-CSF during induction yielded disappointing results. The EORTC LCG is currently studying the relative value of various intercalating agents during induction, and of the G-CSF as well, and, during remission the autologous peripheral stem cells compared to bone marrow transplantation.European Organisation of Research and Treatment of Cancer     

Platelet-derived growth factor (PDGF) in human acute myelogenous leukemia: PDGF receptor expression, endogenous PDGF release and responsiveness to exogenous PDGF isoforms by in vitro cultured acute myelogenous leukemia blasts

We investigated effects of Platelet-derived growth factor (PDGF) and Platelet factor 4 (PF-4) on the functional characteristics of native, human acute myelogenous leukemia (AML) blasts. AML blast expression of the PDGF-receptor alpha-chain was detected for a subset of patients (45%), whereas PDGF-receptor beta-chain expression was detected for most patients (90%). Constitutive AML blast release of the PDGF-AB isoform (the major form also derived from normal platelets) was detected for 43% of patients, whereas PDGF-BB release was not detected for any patient. The PDGF isoforms AA, AB and BB had dose-dependent and divergent effects on spontaneous and cytokine-dependent AML blast proliferation, whereas for constitutive cytokine secretion (IL-1beta, IL-6, TNF-alpha) inhibitory effects were rare and all three isoforms usually had no effect or enhanced the constitutive secretion. The PDGF effects were caused by a direct effect on the AML blasts and were not dependent on the presence of serum. The PDGF effects could also be detected after in vitro culture of AML cells in the presence of IL-4+ granulocyte-macrophage colony stimulating factor. PF-4 had divergent effects on proliferation and cytokine secretion by native AML blasts. Our results suggest that exogenous (e.g. platelet-secreted) PDGF and PF-4 can function as regulators of leukemic hematopoiesis and possibly also modulate the function of residual AML cells in peripheral blood stem cell grafts. On the other hand, endogenous release of PDGF-AB by native blasts may modulate the function of normal cells in the bone marrow microenvironment (e.g. bone marrow stromal cells).     

Effects of angiogenic regulators on in vitro proliferation and cytokine secretion by native human acute myelogenous leukemia blasts

Angiogenesis seems to be important in the pathogenesis of acute myelogenous leukemia (AML). The endothelial cell proliferation and microvessel formation are regulated by a wide range of soluble mediators, including angiogenin, angiopoietin-2, basic fibroblast growth factors, vascular endothelial growth factor (VEGF), VEGF-D, angiostatin and endostatin. In the present study, it has been investigated whether these mediators have an additional direct effect on the proliferation and cytokine release by native human AML blasts. AML cells derived from a large group of consecutive patients were investigated. All these mediators could alter the proliferation and cytokine release [interleukin (IL) 1beta, IL6, IL8, tumor necrosis factor alpha] for a minority of patients. Alteration of spontaneous proliferation by at least one mediator was detected in five of 38 patients; whereas, altered cytokine (Flt3-ligand, granulocyte-macrophage colony-stimulating factor, stem cell factor)-dependant proliferation was observed for 10 patients. Growth enhancement was most frequently observed, whereas growth inhibition was uncommon. The effects on AML blast proliferation were often dependant on or were modulated by the presence of the three hematopoietic growth factors. Based on the present results, it is concluded that angioregulatory mediators have additional growth-enhancing effects directly on the AML blasts for certain patients. However, based on the results from this investigation and previous studies it is suggested that their major contribution to the pathogenesis of AML is through their effects on regulation of bone marrow angiogenesis, and future studies of these mediators in AML should probably focus on these effects.     

Cutis verticis gyrata: Unusual chloromatous disease in acute myelogenous leukemia

Chloromas are an unusual finding in patients with acute or chronic granulocytic leukemia. The present report describes a man with acute myelogenous leukemia (AML) whose course was complicated by cutis verticis gyrata (CVG), a mediastinal mass, neuropathies, and hepatosplenomegaly as a result of visceral involvement. Leukemic tumors and organ infiltration developed independent of his hematologic status and resulted in significant morbidity and, eventually, mortality from gastrointestinal hemorrhage. Nevertheless, at least temporary control over these complications was achieved when they were managed as leukemic “sanctuaries” and treated with local irradiation.     

Effects of arsenic trioxide (As2O3) on leukemic cells from patients with non-M3 acute myelogenous leukemia: studies of cytotoxicity, apoptosis and the pattern of resistance

Arsenic oxide (As2O3) has recently been reported to induce remission in a high percentage of patients with acute promyelocytic leukemia (APL). The aim of this study was to investigate the effects of As2O3 at therapeutic concentrations on cell viability and apoptosis on leukemic cells from patients with non-M3 acute myelogenous leukemia (AML) and to study the resistance profile compared to conventional AML drugs. Cells from 20 patients were exposed to therapeutic concentrations of As2O3 continuously for 96 h. As2O3 reduced the viability in blast cells from all the 20 tested patients compared to unexposed controls (p-value: 0.02 at 0.05 microM; <0.005 at 1.0 microM and higher concentrations). An increase in the apoptotic rate was also seen after incubation with As2O3. Parallel to the incubation with arsenic the in vitro sensitivity to a number of chemotherapeutic agents commonly used in AML was studied. Correlation coefficients for the in vitro sensitivity were highly significant between the conventional AML drugs except for Ara-C. For As2O3, all the correlation coefficients were negative and ranged between -0.05 and -0.51. Furthermore, increased P-gp expression in a multidrug resistant HL-60 cell line did not decrease the sensitivity to As2O3 as compared to the parental cell line. Neither did a P-gp-transfected variant of the K562 cell line show decreased sensitivity to As2O3. We conclude that As2O3 at therapeutic concentrations induces apoptosis and cytotoxic effects in blast cells from patients with non-M3 AML, and that As2O3 differs from conventional AML drugs with respect to the mechanisms that confer resistance to the drugs.     

Extensive neoplastic cardiac infiltration in a patient with acute myelogenous leukemia: role of echocardiography

We present the case of a 34-year-old man with a history of relapsed acute myelogenous leukemia with extensive metastatic infiltration of the endomyocardium. Transesophageal echocardiography revealed masses within all the cardiac chambers and associated vascular structures. Biopsy of the superior vena cava mass had the histological features of a granulocytic sarcoma (GS, chloroma). Massive involvement of the heart with GS is very rare, and only a few reports have been published in the literature.     

A family inheriting different subtypes of acute myelogenous leukemia

Rare inherited cancer syndromes have proven invaluable for the identification of genes involved in the more frequent corresponding noninherited cases. We report on a family with an adult onset, incompletely penetrant, autosomal dominant syndrome of myelodysplasia and acute myelogenous leukemia, affecting at least eight, and probably ten, individuals from three generations. The patients have developed leukemias differing in morphologic subtype, tumor cytogenetics, and abruptness of presentation. Some have presented with acute onset and others with protracted myelodysplasia. This family does not have an unusual incidence of other malignancies; however, one person at 50% risk of inheriting this gene developed atypical mycobacterium infection in the absence of leukemia, but also without appreciable risk factors for acquired deficiencies in cellular immunity. Features common to affected family members, including the individual with mycobacterium infection, are the early presence in the bone marrow of red cell and platelet maturation defects. A search for mutations in diseased marrows fails to detect abnormalities of p53 or N-ras. Two of the affected family members, third degree relatives, have co-inherited a constitutional chromosomal banding variation of 9p21–22, potentially suggesting linkage to this locus. The variable penetrance and expressivity of this syndrome support a multistep model of leukemia evolution, in which the gene defined by this family's syndrome is the signal step. © 1996 Wiley-Liss, Inc.     

Calcium ionophore: a single reagent for the differentiation of primary human acute myelogenous leukaemia cells towards dendritic cells

Blood monocytes and CD34(+) haemopoietic progenitor cells, as well as certain leukaemic cell lines, acquire characteristics of mature dendritic cells (DC) after stimulation with calcium ionophore (CI). We studied whether the in vitro treatment of primary human acute myelogenous leukaemia (AML) cells with CI leads to differentiation towards DC. Blast cells derived from nine AML patients were cultured in the presence of either CI or an established differentiation cocktail consisting of granulocyte-macrophage colony-stimulating factor plus interleukin 4 and tumour necrosis factor-alpha for 5-7 d. Microscopic examination revealed that under both conditions, AML cells were shifted along the DC pathway. In seven out of nine cases, CI-cultivation led to a higher proportion of cells with dendritic morphology. The percentage of CD40 and CD86 expressing cells was significantly increased upon CI treatment compared with cytokine-cultured cells. DC molecules as CD80 and CD83 were up-regulated upon calcium mobilization of AML cells in four out of nine samples. In four cases, CI-treated stimulator cells induced an enhanced proliferative allogeneic T-cell response compared with cytokine-treated stimulator cells. In conclusion, these data demonstrate that CI treatment is an alternative in vitro strategy to differentiate human AML cells into DC.     

Chromosome changes in a patient achieving complete remission in the acute phase of chronic myelogenous leukemia.

A patient with chronic myelogenous leukemia (CML) had a Philadelphia chromosome (Ph′) [t(9q+;22q–)] in all cells and trisomy C in 13% of cells (later determined to be trisomy 8) at the time of diagnosis. After 21 months of intermittent treatment with busulfan, the acute phase developed with the appearance of an additional abnormality (trisomy ?19). During a complete remission and reconversion to the chronic phase, trisomies 8 and ?19 disappeared, although the Ph′ remained. Following a period of marked thrombocytosis, a second relapse occurred with the reappearance of both marker chromosomes.